Gold nanocluster composition and method for detecting thiol-containing compounds

ABSTRACT

A gold nanocluster composition and method for preparing the same are provided. The method includes providing a gold ion-containing solution. Next, the method entails mixing the gold ion-containing solution and a reducing agent solution to obtain a first mixture liquid, and heating the first mixture liquid to obtain a second mixture liquid, wherein the second mixture liquid contains the gold nanoclusters, which are partially capped by reducing agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of pending U.S. patent applicationSer. No. 14/582,428, filed on Dec. 24, 2014 and entitled “Goldnanocluster composition and method for preparing the same and method fordetecting thiol-containing compounds”, which is based on, and claimspriority from, Taiwan Application Serial Number 103138484, filed on Nov.6, 2014, the disclosure of which is hereby incorporated by referenceherein in its entirety.

TECHNICAL FIELD

The technical field relates to a gold nanocluster composition and amethod for preparing the same, and in particular it relates to a methodutilizing a gold nanocluster composition for detecting athiol-containing compound.

BACKGROUND

Recent research shows that when the size of the gold nanoparticles isreduced to that of the gold nanoclusters, they have specific fluorescentproperties other than surface plasmon resonance. Because the goldnanoclusters have advantages such as a lower toxicity than thesemiconductor fluorescence quantum dots and high light stability, thegold nanoclusters have great potential in the applications of biosensingand bioluminescence labeling. However, conventional methods forsynthesizing the gold nanoclusters and modifying their surface arecomplicated and time-consuming. If bio-molecules should be conjugated onthe gold nanoclusters, the more complicated and time-consuming processescannot be omitted.

Accordingly, a process for easy preparation and surface modification ofthe gold nanoclusters is desired in the industry. Furthermore, the goldnanoclusters may utilize for detecting thiol-containing compounds.

SUMMARY

One embodiment of the disclosure provides a gold nanoclustercomposition, comprising: a gold nanocluster partially capped by reducingagent, and the gold nanocluster composition has fluorescence emissionpeaks at wavelength of 600-650 nm and 800-850 nm.

One embodiment of the disclosure provides a gold nanoclustercomposition, comprising: a gold nanocluster partially capped by reducingagent, and the gold nanocluster composition has a single fluorescenceemission peak at wavelength of 800-900 nm.

One embodiment of the disclosure provides a method for preparing a goldnanocluster composition, comprising: mixing a gold ion-containingsolution and a reducing agent solution to obtain a first mixture liquid;and heating the first mixture liquid to obtain a second mixture liquid,wherein the second mixture liquid contains a gold nanoclustercomposition, and the gold nanoclusters are partially capped by reducingagent.

One embodiment of the disclosure provides a method for detecting athiol-containing compound, including: providing the described goldnanocluster compositions; providing an analyte to react with the goldnanocluster compositions; and analyzing the reaction result to checkwhether the analyte contains a thiol-containing compound or not.

A detailed description is given in the following embodiments withreference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure can be more fully understood by reading the subsequentdetailed description and examples with references made to theaccompanying drawings, wherein:

FIG. 1 shows a flow chart of preparing a gold nanocluster composition inone embodiment of the disclosure;

FIG. 2 shows the gold nanocluster compositions are partially capped byglutathione in one embodiment of the disclosure;

FIGS. 3A-3B, 4, 5, and 6 show the specific fluorescence spectra of thegold nanocluster compositions prepared at different molar ratios of goldions to glutathione in embodiments of the disclosure;

FIGS. 7A-7E and 8A-8E show the fluorescence spectra of the goldnanocluster compositions (prepared at different molar ratios of goldions to glutathione) detecting thiol-containing compounds from liquidanalytes in embodiments of the disclosure;

FIGS. 9A-9D, 10A-10D, and 11A-11D show the change of fluorescenceintensity of the gold nanocluster compositions (prepared at differentmolar ratios of gold ions to glutathione) detecting thiol-containingcompounds from liquid analytes in embodiments of the disclosure;

FIG. 12 shows the fluorescence spectrum obtained from the mixturesolution of the gold nanocluster compositions and water (or glutathioneaqueous solution) in one embodiment of the disclosure;

FIG. 13 shows the change of fluorescence intensity obtained from themixture solution of the gold nanocluster compositions and liquidanalytes with or without thiol-containing compounds in one embodiment ofthe disclosure; and

FIG. 14 shows the fluorescence spectra obtained from the mixturesolution of the gold nanocluster compositions and gaseous analytes withor without thiol-containing compounds in one embodiment of thedisclosure;

DETAILED DESCRIPTION

In the following detailed description, for purposes of explanation,numerous specific details are set forth in order to provide a thoroughunderstanding of the disclosed embodiments. It will be apparent,however, that one or more embodiments may be practiced without thesespecific details. In other instances, well-known structures and devicesare schematically shown in order to simplify the drawing.

FIG. 1 shows a flow chart of preparing a gold nanocluster composition inone embodiment of the disclosure. First, a gold ion-containing solutionand a reducing agent solution are mixed to form a first mixture liquidin step 11. In one embodiment, the gold ion-containing solution can bechloroauric acid solution, auric chloride solution, gold sulfidesolution, or a combination thereof. The reducing agent can beglutathione (GSH). When the gold ions and the reducing agent have amolar ratio of 1:0.9 to 1:1.4, a gold nanocluster composition formedfrom that may simultaneously have dual fluorescence emission peaks, e.g.at wavelength of 600-650 nm and 800-850 nm. When the gold ions and thereducing agent have a molar ratio of 1:0 to 1:0.6, a mixing liquidformed from that has no fluorescence. When the gold ions and thereducing agent have a molar ratio of 1:1.5 to 1:2, a gold nanoclustercomposition formed from that has deformed fluorescence emission peaks.The fluorescence emission at wavelength of 700 nm of a gold nanoclustercomposition will be weakened by increasing the molar ratio of thereducing agent.

Subsequently, the first mixture liquid is heated to form a secondmixture liquid in step 12. In one embodiment, the heating step can beperformed by a general heating method such as with a dry bath heater ora microwave heater. In one embodiment, the heating step is performed bymicrowave power of 270 W to 450 W for a period of 10 minutes to 60minutes. The gold ions cannot completely react to form a goldnanocluster composition by overly low microwave power or an overly shortheating period. The gold ions easily form larger gold nanoparticles byoverly high microwave power or an overly long heating period. The secondmixture liquid formed by the heating process contains the goldnanocluster compositions, wherein the gold nanoclusters are partiallycapped by reducing agent. The term “partially” means that not the entiresurface of each of the gold nanoclusters is fully capped by reducingagent, and some unoccupied sites on the surface of the gold nanoclustersremain.

Finally, the second mixture liquid can be centrifuged in step 13 tocollect the supernatant for obtaining the gold nanocluster compositions.The solution containing gold nanocluster compositions is stored at 4° C.for further use. In some embodiments, the rotation speed of thecentrifugal step is 10000 rpm to 14000 rpm. In another embodiment, thefactors of the centrifugal step such as rotation speed, rotation period,and rotation frequency are not limited. Only if the gold nanoclustercompositions can be separated by a set of centrifugal factors, the setof factors are accepted.

In another embodiment, the steps 11 to 13 are repeated, the differencebeing that the gold ions and the reducing agent have a molar ratio of1:0.7 to 1:0.8, and the other process factors of the heating step andthe centrifugal step are similar to the above embodiment. As such, thegold nanoclusters are partially capped by reducing agent, and the goldnanocluster compositions have a single fluorescence emission peak atwavelength of 800 nm to 900 nm.

The fluorescent gold nanocluster compositions may serve as signalmolecules due to their specific optical properties. The gold nanoclustercompositions allow for easy modification for different applications,such as biomedicine (e.g. detection, imaging, and drug release therapy).The gold nanocluster compositions with the specific emission propertiescan be applied as a novel optoelectronic material or sensor forenvironmental safety, food safety, and elsewhere in the food industry.

One embodiment of the disclosure provides a method of detecting athiol-containing compound, including: providing the gold nanoclustercompositions to be reacted with an analyte, and analyzing the reactionresult to check whether the analyte contains a thiol-containing compoundor not. In one embodiment, the analyte is in liquid form or gas form.The mixture of the gold nanocluster compositions and the analyte can beimmediately characterized by a fluorescence spectroscopy analysis systemfor measuring the emission intensity at a specific wavelength. Forexample, the specific wavelength is 600-650 nm and 800-850 nm. While theanalyte contains a thiol-containing compound, the fluorescence emissionintensity at wavelength of 600-650 nm increases, and the fluorescenceemission intensity at wavelength of 800-850 nm decreases.

Below, exemplary embodiments will be described in detail with referenceto accompanying drawings so as to be easily realized by a person havingordinary knowledge in the art. The inventive concept may be embodied invarious forms without being limited to the exemplary embodiments setforth herein. Descriptions of well-known parts are omitted for clarity,and like reference numerals refer to like elements throughout.

EXAMPLES

Preparation of the gold nanocluster compositions

Example 1

5 mM chloroauric acid (HAuCl₄) solution and 5 mM glutathione(L-glutathione, GSH) solution were prepared. 0.2 mL of HAuCl₄ solutionand 0.2 mL of GSH solution were added to a microcentrifuge tube, whereinthe molar ratio of HAuCl₄ and GSH was 1:1. The tube was then completelyshaken for 5 minutes using a vortex mixer to obtain a first mixtureliquid. The microcentrifuge tube was opened and then put into a domesticmicrowave oven, heated by microwave power of 270 W for 30 minutes, andthen heated by microwave power of 450 W for 30 minutes to obtain asecond mixture liquid. The microcentrifuge tube was cooled to roomtemperature and then centrifuged at 12000 rpm for 10 minutes. Thesupernatant was collected to obtain a liquid containing the goldnanocluster compositions. Concentrations of HAuCl₄ solution and GSHsolution could be controlled to tune the fluorescence emission peakrange of the gold nanocluster compositions. As shown in FIG. 2, the goldnanoclusters (20) are partially capped by GSH (21).

Example 2

5 mM HAuCl₄ solution and 5 mM GSH solution were prepared. 0.2 mL ofHAuCl₄ solution and 0.2 mL of GSH solution were added to a glass samplevial, wherein the molar ratio of HAuCl₄ and GSH was 1:1. The tube wasthen completely shaken for 10 seconds using a vortex mixer to obtain afirst mixture liquid. The glass sample via was opened and then put intoa dry bath heater, heated from room temperature to 120° C. for 10minutes, and then heated at 120° C. for 50 minutes to obtain a secondmixture liquid. The glass sample vial was cooled to room temperature andthen the solution was centrifuged at 12000 rpm for 10 minutes. Thesupernatant was collected to obtain a liquid containing the goldnanocluster compositions.

Example 3

Example 3 was similar to Example 1, and the difference in Example 3 wasthe concentration of GSH solution being changed to achieve differentmolar ratios of HAuCl₄ and GSH, such as Au:GSH=1:1.1, 1:1.2, 1:1.3,1:1.4, 1:1.5, and 1:1.6. When the Au:GSH was 1:1.1, the gold nanoclustercompositions still had dual fluorescence emission peaks. When the Au:GSHwas 1:1.2, the fluorescence emission peaks of the gold nanoclustercompositions began to deform as shown in FIGS. 3A-3B.

Example 4

Example 4 was similar to Example 2, and the difference in Example 4 wasthe concentration of GSH solution being changed to achieve differentmolar ratios of HAuCl₄ and GSH, such as Au:GSH=1:1.1, 1:1.2, 1:1.3, and1:1.4. The gold nanocluster compositions had dual fluorescence emissionpeaks, as shown in FIG. 4.

Example 5

Example 5 was similar to Example 1, and the difference in Example 5 wasthe concentration of GSH solution being changed to achieve a differentmolar ratio of HAuCl₄ and GSH, such as Au:GSH=1:0.8. The fluorescenceemission spectrum of the gold nanocluster compositions is shown in FIG.3A. Note that the gold nanocluster compositions prepared by above Au:GSHmolar ratio only has a single fluorescence peak at the near-infraredregion with a wavelength greater than 800 nm, other than dualfluorescence emission peaks at wavelength of 600-650 nm and 800-850 nm.

Comparative Example 1

Comparative Example 1 was similar to Example 1, and the difference inComparative Example 1 was the concentration of GSH solution beingchanged to achieve different molar ratios of HAuCl₄ and GSH, such asAu:GSH=1:0, 1:0.1, 1:0.2, 1:0.4, and 1:0.6. The products prepared fromthe above Au:GSH molar ratios had no fluorescent properties, as shown inFIG. 5.

Comparative Example 2

Comparative Example 2 was similar to Example 1, and the difference inComparative Example 2 was the concentration of GSH solution beingchanged to achieve different molar ratios of HAuCl₄ and GSH, such asAu:GSH=1:1.7, 1:1.8, 1:1.9, and 1:2. The products prepared from theabove Au:GSH molar ratios had only one fluorescence emission peak aroundwavelength of 700 nm. The emission intensities of the products weregradually decreased by increasing the GSH molar ratios, as shown in FIG.6.

Proof of the gold nanoclusters are partially capped by GSH 15 μL of GSHsolutions of different concentrations (0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM,0.75 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, and 4 mM) were added to 15 μLof the products in Comparative Example 1 (Au:GSH=1:0, 1:0.1, 1:0.2,1:0.4, and 1:0.6) in the microwell plate, and then evenly mixed for 1minute. The microwell plate was put into a fluorescence spectroscopyanalysis system, and then excited by a light beam with wavelength of 365nm to analyze the fluorescent properties of the mixtures. As shown inFIGS. 7A-7E, the products in Comparative Example 1 had no obviousfluorescence emission change after adding GSH solutions.

15 μL of GSH solutions of different concentrations (0.05 mM, 0.1 mM,0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, and 4 mM)were added to 15 μL of the liquid products in Example 1 (Au:GSH=1:1) andExample 3 (Au:GSH=1:1.1, 1:1.2, 1:1.3, and 1:1.4) in the microwellplate, and then evenly mixed for 1 minute. The microwell plate was putinto a fluorescence spectroscopy analysis system, and then excited by alight beam with wavelength of 365 nm to analyze the fluorescentproperties of the mixtures. As shown in FIGS. 8A-8E, the goldnanocluster compositions in Examples 1 and 3 had enhanced fluorescenceintensities at wavelength of 600-650 nm and weakened fluorescenceintensities at wavelength of 800-850 nm after adding GSH solutions.Especially when the Au:GSH molar ratios were 1:1, 1:1.1, and 1:1.2 forpreparing the gold nanocluster compositions, the intensities offluorescence emission peak significantly changed. Since the fluorescenceof gold nanoclusters originates from the charge transfer between theligands and gold nanocluster core through the Au—S bonds, as inferredfrom the above result, the gold nanoclusters were partially capped byGSH. Therefore, the gold nanocluster compositions still had unoccupiedsites for further bonding to additional GSH, thereby changing thefluorescence emission intensities thereof

When the Au:GSH molar ratios were 1:1.3 to 1:1.4, the products haddeformed and shifted fluorescence emission spectra with minor intensitychanges after adding 15 μL of GSH solutions of different concentrations(0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM,3 mM, and 4 mM). As inferred from the above result, using higher GSHmolar ratio for preparing the gold nanocluster compositions resulted inmore GSH bonded on the surface of the gold nanoclusters. Therefore,fewer unoccupied sites on the surface of the gold nanoclusters could bebonded to the further added GSH.

Analysis of the fluorescence emission peak intensity of the goldnanocluster compositions after adding further GSH

The sum of the increase of the fluorescence emission intensity atwavelength of 630 nm and the decrease of the fluorescence emissionintensity at wavelength of 810 nm was defined as ΔRFU for revealing therelation between the change of fluorescence emission intensity and theconcentration of GSH. While the Au:GSH molar ratios were 1:0.8 to 1:1.2,the ΔRFUs as a function of the concentration of the additional added GSHare shown in FIGS. 9A-9D. Because GSH simultaneously served as thereducing agent and the capping agent, as inferred from the above result,lower GSH concentration used during the synthesis tend to form largergold nanocluster compositions with lower amounts of Au (I) on thesurface for bonding to thiols. Thereby causing fewer change of thefluorescence emission intensity after adding additional GSH. The higherGSH concentration used during the synthesis should form smaller goldnanocluster compositions with higher amounts of Au (I) on the surfacefor bonding to thiols. As such, the fluorescence emission intensitychanged significantly after adding additional thiols.

In another example, the Au:GSH molar ratios were 1:1.3 to 1:1.6, suchthat the surface of the gold nanoclusters was capped by more GSH. Assuch, their fluorescence emission peak intensity changes were less andsaturated earlier after adding additional GSH, as shown in FIGS.10A-10D.

In a further example, the Au:GSH molar ratios were 1:1.7 to 1:1.9, suchthat the surface of the gold nanoclusters was capped by more GSH. Assuch, their fluorescence emission peak intensity changes were less andsaturated earlier after adding additional GSH, as shown in FIGS.11A-11C. FIG. 11D shows a comparison of the Au:GSH molar ratios of1:1.2, 1:1.4, and 1:1.7.

Referring to the synthesis of the disclosure with Au:GSH molar ratios of1:0.8 to 1:1.2, the gold nanocluster compositions prepared by more GSHcould have enhanced fluorescence emission peaks at wavelength of 600-650nm and weakened fluorescence emission peaks at wavelength of 800-850 nm,the similar behavior as adding additional GSH. When the gold nanoclustercompositions were prepared from a higher GSH concentration, theirfluorescence emission peak intensity changes were less and saturatedearlier. As a result, the Au:GSH molar ratio can be controlled to tunethe amount of GSH capped on the surface of the gold nanoclusters.

Furthermore, the fluorescence emission spectra of the gold nanoclustercompositions changed after reacting with additional added GSH. Forexample, the fluorescence intensity at wavelength of 630 nm wasenhanced, and the fluorescence intensity at wavelength of 810 nm wasweakened. Accordingly, the gold nanocluster compositions might serve assensors for thiol analytes.

Although the above fluorescence emission peak changes could bedetermined by fluorescence spectra, the fluorescence emission intensitychanges could also be determined by naked eye.

Detection of an Aqueous Solution of Thiol-Containing Analytes

First, 0.2 mM of aqueous solution of different analytes were prepared inmicrocentrifuge tubes. Sample 1 was water, Sample 2 was a glycinesolution, Sample 3 was a glutamic acid solution, Sample 4 was ahistidine solution, Sample 5 was a methionine solution, Sample 6 was acystine solution, Sample 7 was an oxidized glutathione solution, Sample8 was a cysteine solution, Sample 9 was an N-acetylcysteine solution,Sample 10 was a penicillamine solution, Sample 11 was an L-glutathionesolution, Sample 12 was a mercaptoethanol solution, and Sample 13 was acysteine-histidine solution. The aqueous solution of the goldnanocluster compositions in Example 3 (Au:GSH=1:1.1) was selected todetect the thiol-containing compounds, and diluted 10 times withde-ionized water for further use.

15 μL of diluted aqueous solution of the gold nanocluster compositionsand 15 μL of a solution of Samples were added to a microcentrifuge tube,respectively, and the tube was then shaken using a vortex mixer for 15minutes to form mixture solutions. The mixture solutions were added tomicrowell plates, respectively, which were put into a fluorescencespectroscopy analysis system, and then excited by a light beam withwavelength of 365 nm to analyze the fluorescent property of themixtures. For example, the fluorescence emission intensity at wavelengthof 615 nm (Fx) and the fluorescence emission intensity at wavelength of815 nm (Fy) of the analytes were recorded. The fluorescence emissionintensity at wavelength of 615 nm (F1x) and the fluorescence emissionintensity at wavelength of 815 nm (F1y) of Sample 1 were set as blank,and the fluorescence emission intensity changes of the samples weredefined as ΔRFU=(Fx−F1x)+(F1y−Fy). The ΔRFU of Samples were shown inFIG. 13.

As shown in FIG. 13, the fluorescence emission intensity of the goldnanocluster compositions would change after mixing with athiol-containing compound, such as Sample 8 (cysteine), Sample 9(N-acetylcysteine), Sample 10 (penicillamine), Sample 11(L-glutathione), Sample 12 (mercaptoethanol), and Sample 13(cysteine-histidine). On the other hand, the fluorescence emissionintensity of the gold nanocluster compositions is nearly unaffectedafter mixing with a compound free of thiol, such as Sample 2 (glycine),Sample 3 (glutamic acid), Sample 4 (histidine), Sample 5 (methionine),Sample 6 (cystine), and Sample 7 (oxidized glutathione). Accordingly,the gold nanocluster compositions of the disclosure were specific to thethiol-containing compounds.

Detection of a Thiol-Containing Gas

The aqueous solution of the gold nanocluster compositions in Example 3(Au:GSH=1:1.1) was selected to detect the thiol-containing gas, anddiluted half with deionized water for further use. 0.3 mL of the dilutedaqueous solution of the gold nanocluster compositions were added to amicrocentrifuge tube. The microcentrifuge tube was opened and fixed in acommercially available airtight container (with a volume of 1.4 L). 1.4μL of ethanol was dropped into the airtight container and then quicklysealed and put in an oven at 40° C., thereby vaporizing the ethanol toform a vapor with a concentration of 1 ppm. After 30 minutes, themicrocentrifuge tube was taken out from the airtight container and theaqueous solution of the gold nanocluster compositions was added to amicrowell plate. The microwell plate was put into a fluorescencespectroscopy analysis system, and then excited by a light beam withwavelength of 365 nm to analyze the fluorescent property of the aqueoussolution. The above steps were repeated again, and the differencethereof was the ethanol being replaced with mercaptoethanol.

As shown in FIG. 14, the aqueous solution contacting the mercaptoethanolvapor had an enhanced fluorescence emission intensity at wavelength of600-650 nm and a weakened fluorescence emission intensity at wavelengthof 800-850 nm. The aqueous solution contacting the ethanol vapor had nofluorescence emission intensity change at wavelength of 600-650 nm and800-850 nm. Accordingly, the gold nanocluster compositions were specificto the mercaptoethanol vapor.

As shown in above experiments, the gold nanocluster compositions in thedisclosure were partially capped by reducing agent. The gold nanoclustercompositions could be obtained by controlling the molar ratio of thegold ions and the reducing agent, such that the gold nanoclustercompositions had dual fluorescence emission peaks at wavelength of600-650 nm and 800-850 nm. A liquid (or a gas) with or without a thiolcompound could be immediately checked by measuring the change of thefluorescence emission spectra of the gold nanocluster compositionsbefore and after contacting the liquid (or the gas).

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the disclosed methods andmaterials. It is intended that the specification and examples beconsidered as exemplary only, with a true scope of the disclosure beingindicated by the following claims and their equivalents.

What is claimed is:
 1. A gold nanocluster composition, comprising: agold nanocluster partially capped by reducing agent, and the goldnanocluster composition has fluorescence emission peaks at wavelength of600-650 nm and 800-850 nm.
 2. The gold nanocluster composition asclaimed in claim 1, wherein the reducing agent comprises glutathione. 3.A gold nanocluster composition, comprising: a gold nanocluster partiallycapped by reducing agent, and the gold nanocluster composition has asingle fluorescence emission peak at wavelength of 800-900 nm.
 4. Thegold nanocluster composition as claimed in claim 3, wherein the reducingagent comprises glutathione.
 5. A method for detecting athiol-containing compound, including: providing the gold nanoclustercompositions as claimed in claim 1; providing an analyte to react withthe gold nanocluster compositions; and analyzing the reaction result tocheck whether the analyte contains a thiol-containing compound or not.6. The method as claimed in claim 5, wherein the analyte is gas orliquid.
 7. The method as claimed in claim 5, wherein the mixture of thegold nanocluster compositions and the analyte can be immediatelycharacterized by a fluorescence spectroscopy analysis system formeasuring the emission intensity at specific wavelengths.
 8. The methodas claimed in claim 7, wherein the specific wavelengths are 600-650 nmand 800-850 nm.
 9. The method as claimed in claim 7, wherein thereaction result has an enhanced emission intensity at wavelength of600-650 nm and a weakened emission intensity at wavelength of 800-850 nmwhen the analyte contains a thiol-containing compound.